PROJECT SUMMARY/ABSTRACT Gram-negative bloodstream infections (BSIs) cause severe morbidity and mortality in neutropenic patients. Fluo- roquinolones (FQs) are used to prevent Gram-negative BSI during neutropenia, but the extent to which FQ resistance threatens the effectiveness of FQ prophylaxis is unknown. The objective of this proposal is to deter- mine how colonization with FQ-resistant Enterobacterales (FQRE) impacts the risk of Gram-negative BSI in neutropenic patients who receive FQ prophylaxis and how FQRE colonization density and gut microbiome di- versity influence this risk. The hypothesis is that the effectiveness of FQ prophylaxis in neutropenic patients is markedly diminished in patients colonized with FQRE, particularly if colonized above a quantitative threshold, and that absence of commensal gut bacteria increases the risk of Gram-negative BSI. The rationale for this proposal is that knowledge of the impact of FQRE colonization and gut microbiome diversity on the effectiveness of FQ prophylaxis could lead to individualized infection prevention strategies. The specific aims of this project are: 1) Determine the prevalence and clinical significance of FQRE colonization in neutropenic hematopoietic cell transplant (HCT) recipients who receive FQ prophylaxis; 2) Identify risk factors for FQRE BSI in FQRE- colonized HCT recipients who receive FQ prophylaxis during neutropenia. This proposal will utilize an estab- lished cohort of 350 HCT recipients who received FQ prophylaxis during neutropenia. Stool samples have been collected upon initiation of chemotherapy and weekly thereafter until recovery from neutropenia. For this pro- posal, these samples will be cultured for FQRE. We will determine the prevalence of and risk factors for FQRE colonization on admission for transplant, and compare the risk of Gram-negative BSI during the transplant ad- mission between patients colonized and not colonized with FQRE. We will then sequence the bloodstream and colonizing FQRE to determine how frequently FQRE-colonized HCT recipients develop BSI from their colonizing strain. We will also perform quantitative cultures for FQRE to determine whether there is a quantitative threshold of FQRE colonization that predisposes to breakthrough BSI. We will then perform 16S rRNA sequencing of stool samples from FQRE-colonized patients, compare the microbiome diversity of patients who do and do not develop FQRE BSI, and identify bacterial taxa that are associated with a lower risk of FQRE BSI. We will then assess whether these variables are independently associated with FQRE BSI in a multivariate model. The expected contribution of this proposal is that we will determine whether screening for and quantifying FQRE colonization, combined with an assessment of gut microbiome diversity, can identify neutropenic patients at high risk of de- veloping Gram-negative BSI despite FQ prophylaxis. This contribution would be significant and innovative be- cause it would set the foundation for designing and evaluating an individualized approach to antibacterial prophy- laxis in neutropenic patients that takes into account the presence and density of FQRE colonization and gut microbiome diversity, instead of the current ?one-size-fits-all? approach.